Issue 2, 2017

Magnetic circular dichroism studies of iron(ii) binding to human calprotectin

Abstract

Calprotectin (CP) is an abundant metal-chelating protein involved in host defense, and the ability of human CP to bind Fe(II) in a calcium-dependent manner was recently discovered. In the present study, near-infrared magnetic circular dichroism spectroscopy is employed to investigate the nature of Fe(II) coordination at the two transition-metal-binding sites of CP that are a His3Asp motif (site 1) and a His6 motif (site 2). Upon the addition of sub-stoichiometric Fe(II), a six-coordinate (6C) Fe(II) center associated with site 2 is preferentially formed in the presence of excess Ca(II). This site exhibits an exceptionally large ligand field (10Dq = 11 045 cm−1) for a non-heme Fe(II) protein. Analysis of CP variants lacking residues of the His6 motif supports that CP coordinates Fe(II) at site 2 by employing six His ligands. In the presence of greater than one equiv. of Fe(II) or upon mutation of the His6 motif, the metal ion also binds at site 1 of CP to form a five-coordinate (5C) Fe(II)–His3Asp motif that was previously unidentified in this system. Notably, the introduction of His-to-Ala mutations at the His6 motif results in a mixture of 6C (site 2) and 5C (site 1) signals in the presence of sub-stoichiometric Fe(II). These results are consistent with a reduced Fe(II)-binding affinity of site 2 as more weakly coordinating water-derived ligands complete the 6C site. In the absence of Ca(II), both sites 1 and 2 are occupied upon addition of sub-stoichiometric Fe(II), and a stronger ligand field is observed for the 5C site. These spectroscopic studies provide further evaluation of a unique non-heme Fe(II)–His6 site for metalloproteins and support the notion that Ca(II) ions influence the Fe(II)-binding properties of CP.

Graphical abstract: Magnetic circular dichroism studies of iron(ii) binding to human calprotectin

Supplementary files

Article information

Article type
Edge Article
Submitted
04 Aug 2016
Accepted
11 Oct 2016
First published
11 Oct 2016
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2017,8, 1369-1377

Magnetic circular dichroism studies of iron(II) binding to human calprotectin

T. M. Baker, T. G. Nakashige, E. M. Nolan and M. L. Neidig, Chem. Sci., 2017, 8, 1369 DOI: 10.1039/C6SC03487J

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